Inhibition of MEK Signaling Attenuates Cancer Stem Cell Activity in Anaplastic Thyroid Cancer.

Background: Anaplastic thyroid cancer (ATC) is highly aggressive and has very limited treatment options. Recent studies suggest that cancer stem cell (CSC) activity in ATC could underlie this recurrence and resistance to treatment. The recent approval by the U.S. Food and Drug Administration of the combined treatment of BRAF and MEK inhibitors for ATC patients has shown some efficacy in patients harboring the BRAFV600E mutation. However, it was unknown whether the combined treatment could affect the CSC activity. This study explores the effects of the BRAF and MEK inhibitors on CSC activity in human ATC cells. Methods: Using three human ATC cells, THJ-11T, THJ-16T, and 8505C cells, we evaluated the effects of dabrafenib (a BRAF kinase inhibitor), trametinib (an MEK inhibitor), or a combined treatment of the two drugs on the CSC activity by tumorsphere formation, Aldefluor assays, expression profiles of key CSC markers, immunohistochemistry, and in vivo xenograft mouse models. Furthermore, we also used confocal imaging to directly visualize the effects on drugs on CSCs by the SORE6-mCherry reporter in cultured cells and xenograft tumor cells. Results: The BRAF inhibitor, dabrafenib, had weak efficacy, while the MEK inhibitor, trametinib, showed strong efficacy in attenuating the CSC activity, as evidenced by suppression of CSC marker expression, tumorsphere formation, and Aldefluor assays. Using ATC cells expressing a fluorescent CSC SORE6 reporter, we showed reduction of CSC activity in the rank order of combined > trametinib > dabrafenib through in vitro and in vivo xenograft models. Molecular analyses showed that suppression of CSC activity by these drugs was, in part, mediated by attenuation of the transcription by dampening the RNA polymerase II activity. Conclusions: Our analyses demonstrated the presence of CSCs in ATC cells. The inhibition of CSC activity by the MEK signaling could partially account for the efficacy of the combined treatment shown in ATC patients. However, our studies also showed that not all CSC activity was totally abolished, which may account for the recurrence observed in ATC patients. Our findings have provided new insights into the molecular basis of efficacy and limitations of these drugs in ATC patients.

Lateral approach contributes to shortened surgical time in video-assisted neck surgery (VANS) for thyroid nodule.

Background: Despite endoscopic thyroid surgery contributing to excellent cosmetic outcomes, it requires longer surgical time than open surgery. This study evaluates the factors associated with operative time in video-assisted neck surgery. Methods: We retrospectively reviewed patients who underwent hemithyroidectomy by video-assisted neck surgery at a single hospital between 2017 and 2021. The clinical parameters were evaluated: age, sex, body weight, body mass index, coexistence of Hashimoto thyroiditis, side of operation, surgical approach to thyroid (midline approach or lateral approach), operation time, amount of blood loss during surgery, maximum tumor diameter and tumor volume, and postoperative complications. These parameters were compared to investigate the association of operative time and surgical approach. Results: Seventy consecutive patients were enrolled in this study, the median age at operation was 50 years, and there were 6 (8.6%) men and 64 (91.4%) women. The median body weight and body mass index at operation were 56.6 kg and 21.5, respectively. Coexistence of Hashimoto thyroiditis was found in 17 (24.3%) patients. Significant differences were found between the groups with longer and shorter operation time than median 201 minutes in surgical approaches to the thyroid (P < .001) and the amount of bleeding during surgery (P = .039). There were no differences in other candidates between the groups. Median operation times were 242 minutes in midline approach and 131 minutes in lateral approach (P < .001). Conclusion: The lateral approach of video-assisted neck surgery to the thyroid can contribute to shortened surgical time in the case of benign thyroid nodules.

A case of concurrent occurrence of carcinoma showing thymus-like differentiation and follicular variant of papillary thyroid cancer in the same thyroid.

Carcinoma showing thymus-like differentiation (CASTLE) is a rare thyroid cancer. This is the first report of a case of concurrent occurrence of CASTLE with papillary thyroid carcinoma (PTC). A 66-year-old male patient had hoarseness with right vocal cord paralysis. Ultrasonography revealed a hypoechoic nodule in the inferior pole of the right thyroid lobe. Ultrasound-guided fine-needle aspiration cytology suggested differentiated thyroid cancer. The patient underwent total thyroidectomy with neck dissection. Pathological examination revealed two different thyroid cancers: a CASTLE and a follicular variant of PTC. Postoperative radiation therapy was performed. The patient was still alive after 5 year following the initial treatment without evidence of recurrence. The oncological management of patients with concurrent occurrence of different thyroid cancers should consider the biological behavior of both tumors.

A Rare Case of Thyroid Carcinoma Showing Thymus-Like Differentiation in a Young Adult.

Thyroid carcinoma showing thymus-like differentiation (CASTLE) is thought to originate from ectopic thymic tissue or remnants of the developing thymus within or adjacent to the thyroid. This case report describes a mass located on the left thyroid of a 28-year-old man. Fine-needle aspiration cytology revealed a number of lymphoid cells without atypia that were similar to those seen in a malignant lymphoma of the thyroid, and surgery was performed. Based on additional histopathological findings, the tumor was finally diagnosed as a CASTLE. It is difficult to diagnose this neoplasm using fine-needle aspiration cytology. However, it is possible to differentially diagnose CASTLE based on its histological features. CD5 is useful for diagnosing CASTLE with immunohistochemical staining.

Synergistic Effects of Lenvatinib (E7080) and MEK Inhibitors against Anaplastic Thyroid Cancer in Preclinical Models.

E7080, known as lenvatinib, is an oral multitargeted tyrosine kinase inhibitor that has been shown to improve the survival rate of patients with radioiodine-refractory thyroid cancer. However, a majority of patients do not continue lenvatinib intake due to disease progression or significant toxicity. To improve treatment success rates, we propose the combination of lenvatinib with mitogen-activated protein kinase (MEK) inhibitors. To test this hypothesis, we tested the effects of lenvatinib with the MEK inhibitor U0126 in vitro using two human anaplastic thyroid cancer (ATC) cell lines, 8505C and TCO1, and with another MEK inhibitor, selumetinib (AZD6244), in an ATC mouse model. We found that the combination of lenvatinib with MEK inhibitors enhanced the antitumor effects of monotherapy with either agent in vitro and in vivo, and these effects may be through the AKT (Protein Kinase B) and extracellular signal-regulated kinase (ERK) signaling pathways. Furthermore, the combination does not have significant adverse effects in the ATC mouse models in terms of body weight, blood biochemical parameters, and histopathology. In conclusion, the combination of lenvatinib with an MEK inhibitor is a potentially viable therapeutic approach for ATC treatment.

Funiculosin variants and phosphorylated derivatives promote innate immune responses via the Toll-like receptor 4/myeloid differentiation factor-2 complex.

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) complex is essential for LPS recognition and induces innate immune responses against Gram-negative bacteria. As activation of TLR4/MD-2 is also critical for the induction of adaptive immune responses, TLR4/MD-2 agonists have been developed as vaccine adjuvants, but their efficacy has not yet been ascertained. Here, we demonstrate that a funiculosin (FNC) variant, FNC-RED, and FNC-RED and FNC derivatives are agonists for both murine and human TLR4/MD-2. FNC-RED induced nuclear factor-κB (NF-κB) activation via murine TLR4/MD-2, whereas FNC had no TLR4/MD-2 stimulatory activity. Biacore analysis revealed that FNC-RED binds to murine TLR4/MD-2 but not murine radioprotective 105 (RP105)/myeloid differentiation factor-1 (MD-1), another LPS sensor. FNC-RED induced CD14-independent expressions of pro-inflammatory cytokines and co-stimulatory molecules in murine macrophages and dendritic cells. In contrast, FNC-RED stimulation was reduced in CD14-dependent LPS responses, including dimerization and internalization of TLR4/MD-2 and IFN-ß expression. FNC-RED-induced IL-12p40 production from murine dendritic cells was dependent on NF-κB but not MAPK pathway. In addition, fetal bovine serum augmented lipid A-induced NF-κB activation but blocked FNC-RED-mediated responses. Two synthetic phosphate group-containing FNC-RED and FNC derivatives, FNC-RED-P01 and FNC-P01, respectively, activated human TLR4/MD-2, unlike FNC-RED. Finally, computational analysis revealed that this species-specific activation by FNC-RED and FNC-RED-P01 resulted from differences in electrostatic surface potentials between murine and human TLR4/MD-2. We conclude that FNC-RED and its synthetic derivative represent a novel category of murine and human TLR4/MD-2 agonist.

5-S-GAD attenuates Fe²+-induced lipid peroxidation and cell death in a neuronal cell model.

Iron accumulation in brain is associated with a number of common neurodegenerative disorders. N-ß-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), a novel catechol derivative, was isolated from adult flesh fly as a defense substance. We examined the effect of 5-S-GAD on Fe²+-induced lipid peroxidation and cell death in PC12 cells. Treatment of PC12 cells with Fe²+ (1-20 µM) for 24 h induced lipid peroxidation and cell death in a dose-dependent manner. Butylated hydroxyanisole and α-tocopherol inhibited Fe²+-induced lipid peroxidation and cell death. 5-S-GAD inhibited Fe²+-induced lipid peroxidation and cell death. 5-S-GAD protected PC12 cells from Fe²+-induced cell death possibly by blocking lipid peroxidation.

Effect of 5-S-GAD on UV-B-induced cataracts in rats.

PURPOSE: 5-S-Glutathionyl-N-beta-alanyl-3,4-dihydroxyphenylalanine (5-S-GAD) is a novel antibacterial substance purified from Sarcophaga peregrina (flesh fly) that has both a radical scavenging activity and antioxidative activity. This is a report of an investigation of the effect of 5-S-GAD (eyedrops) on UVB-induced cataracts in rats. METHODS: Brown Norway male rats (n = 32; 7 weeks old) were treated with either 5-S-GAD 0.1%, 5-SGAD 1%, astaxanthin (AST) 0.1% suspension eyedrops or the vehicle alone (the solution without 5-S-GAD) three times a day (three doses at 5-min intervals each time). The treatment was scheduled 2 days before UV-B exposure and 2 days after UV-B exposure. Exposure to 100-200 mJ/cm(2) UV-B was performed once a week between drug treatments for 9 consecutive weeks, with a total dose of 1200 mJ/cm(2) UV-B. Ocular penetration of 5-S-GAD was analyzed using high-pressure liquid chromatography (HPLC). Cataract formation was documented by an anterior eye segment analysis system once a week under mydriasis. The light-scattering intensity (LSI) of the anterior superficial cortex region was measured. RESULTS: In the eighth to ninth week after the start of UV-B exposure, the LSI of anterior subcapsular lenses of 5-S-GAD-treated groups, as detected by HPLC, was significantly lower (P < 0.05) than that of the control, whereas no such difference was found in the AST-treated group. CONCLUSION: 5-S-GAD eyedrop application may delay the progression of UV-B-induced cataract in rats.

A novel neuroprotective role of a small peptide from flesh fly, 5-S-GAD in the rat retina in vivo.

N-beta-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an antibacterial substance isolated from flesh fly, has been described as having multipotential biological activities toward various tissues. However, there has been no report testing its action on neural cells. In the present study, we investigate whether 5-S-GAD is neurotoxic or neuroprotective to the rat retina. 5-S-GAD at high doses (more than 200 pmol) induced apoptosis of retinal neurons 7 days after intraocular injection. 5-S-GAD at low doses (2-20 pmol) significantly attenuated the loss of retinal ganglion cells (RGCs) and the thinning of inner retina induced by NMDA in a dose-dependent manner. To understand the protective mechanism of 5-S-GAD, we investigated the influence of 5-S-GAD on the cell survival molecules, phospho-Akt and Bcl-2. 5-S-GAD (2-20 pmol) rapidly increased phospho-Akt expression 1-7 days and Bcl-2 expression 3-7 days after injection. The cellular localization of this increase was both in bipolar cells and RGCs. This neurosurvival effect of 5-S-GAD was further tested using another model of optic nerve injury. 5-S-GAD significantly blocked the apoptosis of RGCs 7 days after optic nerve crush. These results show that 5-S-GAD (2-20 pmol) protects against the NMDA- and optic nerve crush-induced apoptosis of RGCs. The neuroprotective action of 5-S-GAD in the retina might be mediated by the cell survival phospho-Akt/Bcl-2 system and offers a therapeutic option to rescue RGCs from various types of excitotoxic disease, such as glaucoma.